This invention relates to the formation of stable branch migration structures and the various applications of these structures.
The displacement of one strand of a double-stranded nucleic acid by another single strand with an identical nucleotide sequence is a well documented aspect of DNA or RNA replication and genetic recombination in vivo. Branch points are found in nucleic acids that are undergoing this kind of strand displacement where two strands compete for base pairing interactions with complementary sequences of a third strand. The movement of branch points along the strands of the nucleic acid, branch migration, does not require the action of specific enzymes or proteins.
The phenomenon of branch migration in vitro in the renatured molecules of terminally repetitious, circularly permuted bacteriophage DNA was first reported by Lee, Davis, and Davidson JMB 48: 1-22 (1970)!. Branched nucleic acid structures suitable for the study of strand displacements can be constructed in vitro using various hybridization conditions.
Branch migration has been exploited to form or resolve DNA-RNA hybrids. In solutions without formamide, a DNA strand will displace RNA from a DNA-RNA hybrid. This reaction is the basis of a homogeneous nucleic acid hybridization assay developed by Vary et al. of Allied Corporation Nuc. Acids Res. (1987) 15, 6883-6897 and U.S. Pat. No. 4,795,701!. This assay involves RNase digestion of the displaced RNA strand, conversion of AMP to ATP and detection of the product of the conversion by chemiluminescence using luciferase. Vary's method is not applicable to DNA cloning.
In concentrated formamide solutions, a DNA strand may be displaced by RNA to form an R-loop see Thomas, M., White, R. L., & Davis, R. W. (1976) Proc. Nat. Acad. Sci., USA 73, 2294-2298!. Conceptually, R-loop formation is analogous to the displacement of one DNA strand from the end of a duplex. Regions of double-stranded DNA can take up complementary RNA sequences to form R-loops under conditions where RNA:DNA hybrids are more stable Casey, J. and Davidson, N. (1977) NAR 4: 1539-1552!. The enrichment of specific DNA sequences has been accomplished using buoyant density sedimentation to select for R-loop structures containing these sequences. The technique of R-loop formation has not been patented. To date, applications of R-looping procedures involve partial denaturation of the target DNA and have not yielded products that can be directly cloned into standard cloning vectors.
D-loop formation that is analogous to R-loop formation can occur between a DNA strand and a superhelical DNA duplex Radding, C. M., Beattie, K. L., Holloman, W. K., & Wiegand, R. C. (1977) J. Mol. Biol. 116, 825-839!. This reaction depends upon the superhelical free energy and will, thus, not take place with linear DNA molecules. No cloning technology based on this observation has been described. D loop formation in superhelical DNA has been used for specific cleavage Corey, D. R., Pei, D. & Schultz, P. G. (1989) J. Am. Chem. Soc. 111, 8523-8525!.
A method has been developed which uses RecA-coated strands to overcome the limitation of D-loop formation to superhelical DNA molecules Rigas, B., Welcher, A. A., Ward, D. C., & Weissman, S. M. (1986) Proc. Natl. Acad. Sci., USA 83, 9591-9595!. Labeling of the single-stranded probe with biotinylated nucleotides facilitates purification of the D-loop products of this reaction by affinity chromatography. DNA hybrids that contain biotinylated nucleotides have lower melting temperatures than unmodified DNA hybrids, i.e., biotin has a destabilizing effect on the helix Langer, et al. (1981) Proc. Natl. Acad. Sci. USA 78: 6633-6637!. In this method D-loop formation requires implementation of a pretreatment step to facilitate D-loop formation. Moreover, this procedure does not result in products that are directly clonable into existing DNA cloning vectors.
A short DNA strand hybridized to a longer DNA strand will also be rapidly displaced by a homologous, but longer, overlapping strand in vitro see Green, C. and Tibbetts, C. (1981) Nuc. Acids Res. 9, 1905-1918!. This observation forms the basis of diagnostic assays for DNA or RNA sequences that are based on branch migration and DNA strand displacement, described in Collins et al. Mol. & Cell. Probes 2: 15-30 (1988)!, Vary et al. Clin. Chem. 32: 1696-1701 (1986)!, U.S. Pat. Nos. 4,766,064 Williams et al. (1988), Allied Corp.!, 4,766,062 Diamond et al (1988), Allied Corp.!, and 4,795,701 Vary et al (1988), Allied Corp.! and European Patents 0167238 A1 Collins et al (1985), Allied Corp.! and 0164876 A1Collins et al. (1985) Allied Corp.!.
In these assays, a partially double-stranded probe complex is prepared with a detectable label on one of the two strands. This probe complex is then incubated with a sample containing target nucleic acids (i.e., double-stranded nucleic acids that are at least partially homologous to the single-stranded portion of the probe complex) under appropriate hybridization conditions. The target nucleic acids hybridize to the single-stranded portion of the probe complex and undergo branch migration to release the labeled probe strand. The amount of labeled strand released is proportional to the amount of target DNA in a sample. Thus, this assay involves the use of a pre-formed partially double-stranded probe complex to promote branch migration and relies upon the transient nature of the branch migrated structure and the total release of the labeled probe strand.
In these assays, the efficiency of the strand displacement reaction could be enhanced by the addition of volume excluding reagents, such as polyethers, or by pretreatment of the target with Rec A proteins. Others have also noted that the Rec A protein promotes branch migration that proceeds unidirectionally in the 3'.fwdarw.5' direction Cox, M. M. & Lehman, I. R. (1981) Proc. Natl. Acad. Sci. USA 78: 6018-6022!. The diagnostic assays developed by the Allied Corp. use pre-formed duplexes to promote displacement and do not relate to the development of genetic engineering techniques or to the stabilization of the branch migration structure.
Thus, the phenomenon of branch migration initiated by the formation of a stable hybrid has been described in the literature. Although the formation of branched or looped structures has been used for identification, purification, and enrichment of DNA sequences, this technique has not been applied to the development of a directly clonable product. Moreover, stabilization of branch migration structures would enhance the efficiency of procedures that involve collection or identification of these entities. Simple methods for preparing stable branch migration structures have not been reported by others.
Experiments described more than 20 years ago showed that the substitution of bromine at position C5 of pyrimidines leads to increased duplex stability Michelson et al.(1967) Prog. Nuc. Acid Res. & Mol. Bio. 6: 84-141!. Radding et al. J. Biol. Chem. 116: 2869-2876 (1962)! showed that dG-BrdC is a more thermally stable base pair than dG-dC. In another study, poly dl:poly BrdC had a melting temperature 26.degree. C. higher than poly dI:poly dC Inman & Baldwin (1964) J. Mol. Bio. 8: 452-469!, and it was further shown that poly BrdC displaced poly dC from a poly dI:poly dC duplex to form a new duplex with poly dI Inman J. Mol. Bio. 9: 624-637 (1964)!. These observations have not been applied to the stabilization of branch migration structures by displacer strands containing modified nucleotide bases.
Tatsumi and Strauss Nuc. Acids Res. 5: 331-347 (1978)! labeled DNA with bromodeoxyuridine (BrdUrd) in vivo in human lymphoid cells and observed a high degree of branch migration after isolation and shearing of the DNA. These workers suggested that this high level of branch migration reflected the increased stability of helices containing BrdUrd and the trapping of the branch migration configuration. Their results further suggest that once formed, halogen-substituted branch migration structures are relatively stable. Tatsumi and Strauss did not investigate the phenomenon of branch migration in vitro, did not use synthetic oligo- or polynucleotides labeled with halogenated nucleotides, i.e., pre-modified displacer strands, in their experiments, and could not use the branch migration structures resulting from their experiments for purposes of cloning or mutagenesis.
Currently, specific DNA fragments derived from genomic DNA are usually identified using Southern blot analysis of the restriction enzyme digestion products of that genomic DNA. Southern blot analysis of DNA is a multi-step procedure that generally involves the use of radioisotopes and autoradiography for fragment identification following electrophoresis, transfer of the electrophoresced products to a nitrocellulose or nylon membrane, and hybridization of the transferred products to sequence-specific DNA probes. Procedures that provide for simultaneous labeling and identification of specific DNA fragments would be significantly simpler, faster, and cheaper than Southern blot analyses and would have a significant impact on the field of genetic engineering.
Labeling of DNA sequences has come to imply the incorporation of modified nucleotides at internal or terminal positions. Often this is accomplished enzymatically using nucleotides labeled with small compounds such as biotin, sulfhydryl groups, mercury, allylamine, and digoxigenin. Enrichment or purification of these labeled nucleic acids can be accomplished by affinity chromatography. For example, biotinylated DNA, including D loops, can be selectively bound and eluted from solid matrices bearing avidin or streptavidin groups, as has been reported by Ward and his co-workers Langer, op. cit.!. Similarly, DNA labeled with sulfhydryls can be purified by affinity chromatography on mercurated agarose and mercurated DNA can be purified based on its affinity for sulfhydryl groups. The enrichment of mRNA from total RNA populations can be accomplished based on the affinity of the polyA tails on the messengers for oligo-dT matrices. Following their purification, these enriched or purified nucleic acid sequences are often further subjected to a series of procedures that render them clonable. Labeling procedures that permit sequence enrichment or purification by affinity chromatography and then the direct cloning of these enriched or purified fractions would have significant advantages over existing techniques.
The identification or enrichment of a specific subset of nucleotide sequences or of DNA fragments in isolated genomic DNA or total populations of RNA for the express purpose of cloning those sequences or fragments has been accomplished by a variety of methods that include, but are not limited to those involving the selection of DNA fragments capable of undergoing branch migration, R-loop, or D-loop formation. Selective cloning of fragments in a population has been achieved based on restriction endonuclease cleavage sites, especially those that recognize a unique site Brown. N. L. & Smith, M. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 3213-3216!. and size Thomas, M., Cameron, J. R. & Davis, R. W. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 4579-4583!. Although these strategies have been used widely and successfully to clone a wide variety of genes, they are not universal and have limited specificity.
Another application of genetic engineering techniques relates to the modification of genetic material so as to add or delete nucleotide bases, such as for therapeutic purposes. Efforts to either replace, inactivate, or modify genetic material are currently in progress. In higher eukaryotic organisms, the approaches taken have, thus far, required that the agents used to bring about these changes be incorporated into various plasmid vectors that contain all or part of various viral genomes. Site-directed gene replacement in lower eukaryotes, such as yeasts, has been accomplished. A serious deterrent to the therapeutic manipulation of human genetic material in vivo relates to the lack of a suitable, benign vector system. The ability to perform targeted delivery and incorporation of genetic material into chromosomal DNA without using a viral vector would represent a major advance in the field of gene therapy.
Green and Tibbetts op. cit.! expressed an interest in using branched DNA structures for in vitro site-directed mutagenesis and, in fact, were able to use stable D-loop structures in superhelical DNA for target deletion mutagenesis Proc. Natl. Acad. Sci. USA 77: 2455-2459 (1980)!. They were unable to achieve this goal with linear target DNA molecules using the branch migrated structures they obtained from in vivo labeling of cells with BrdUrd (see Green and Tibbetts, 1981, op. cit.) due to the short half-lives of their branched structures.
Site-specific genetic manipulation has been described Capecchi, M. R. (1989) Science 244, 1288-1292! where a small proportion of the DNA which becomes integrated into the host genome is directed, by homologous recombination, to the desired target DNA. Unfortunately, the additional integration events occur at random and may inactivate or activate genes leading to deleterious consequences. Sequences of DNA capable of initiating triple-helix formation have been reported. Francois, J. C., Saison-Behmoaras, T., Thoung, N. T. & Helene, V. (1989) Biochemistry 28, 9617-9619; Povsic, T. J. & Dervan, P. B. (1989) J. Am. Chem. Soc. 111, 3059-3061!. The utilization of triple helix formation as an adjunct to site-specific genetic manipulation is unknown in the art.